PROJECT SUMMARY Cutaneous wound repair is an essential regenerative process that restores the skin barrier function in response to injury. Impaired wound healing is an important clinical problem that significantly impacts the quality of life of patients and health care cost. Therefore, understanding the molecular control of the wound healing process is crucial to the development of therapeutic agents. Work from our previous funding cycle identified LCN2`s crucial role in cell migration and wound healing. Based on our preliminary data, we hypothesize LCN2 acts through MMP-9 to promote keratinocyte migration and wound healing in a time dependent manner and that manipulating their levels improves wound healing in normal and diabetic mice. We will test our hypothesis with the following specific aims. Aim 1: To determine the mechanism(s) by which LCN2 promotes cell migration and wound healing We will test how inhibition and genetic loss-of-function of MMP9 affects LCN2 regulation of migration and wound repair in vitro and in vivo. Conversely, we will evaluate how exogenous MMP-9 can rescue impaired wound healing in Lcn2-deficient mice. Furthermore, to identify downstream effectors of LCN2, we will employ unbiased comparative proteomics of recombinant LCN2- and control-treated keratinocytes as well as Lcn2 knockout and wild-type-derived keratinocytes. Aim 2: To determine the temporal requirement of LCN2 and MMP-9 in wound healing We hypothesize that LCN2 and MMP-9 are required in the initial phase of wound healing but later need to be downregulated for proper healing to occur. We will manipulate LCN2 and MMP-9 levels at all consecutive key phases during wound repair and establish the timing for an optimal wound healing response as a prerequisite for potential future therapeutic approaches. Aim 3: To determine the effect of LCN2 on wound healing in diabetic mice We will evaluate the effect of topical LCN2 treatment on wound healing in two established diabetic mouse models, and directly compare its response to becaplermin, currently the only FDA-approved topical agent. In addition, we will also evaluate how manipulating LCN2 and MMP-9 in a temporal manner affects diabetic wound healing.